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为了获得同时识别人类和小鼠MAIT细胞的特异性抗体,我们制备了MAIT细胞TCRα链Jα33融合蛋白及其抗体,为MAIT细胞在炎症性肠道疾病(IBD)中作用的研究提供有利的工具。用Jα33的全长序列为引物经PCR扩增Jα33串联体,扩增的串联体片段连接进入T载体,阳性克隆转入pGEX-4T-1表达载体中原核表达GST-Jα33融合蛋白,纯化后的融合蛋白免疫新西兰大白兔制备anti-Jα33多克隆抗体。并分别用Western blot和RT-PCR检测IBD模型小鼠MAIT细胞Jα33的表达。成功获得分子量约为38 kD的融合蛋白,免疫大白兔后得到抗Jα33的多克隆抗体,Western blot结果显示Jα33多克隆抗体能检测到人外周血单个核淋巴细胞表面Jα33的表达,而HeLa中则无Jα33的表达;MAIT细胞Jα33在IBD小鼠肠组织中表达量明显低于正常对照。成功制备了能同时识别人类和小鼠MAIT细胞Jα33的多克隆抗体,为研究MAIT细胞的定位和功能开启了方便之门。
In order to obtain antibodies specifically recognize human and mouse while the MAIT cells, we Jα33 the MAIT cells TCRα chain fusion proteins and antibodies can be prepared to provide an advantageous tool in the study of the role of MAIT cells in inflammatory bowel disease (IBD). Jα33 was amplified by PCR using the full-length sequence of Jα33 as a primer. The amplified tandem fragment was ligated into T vector and the positive clone was transformed into pGEX-4T-1 expression vector to express GST-Jα33 fusion protein. The fusion protein was used to immunize New Zealand rabbits to prepare anti-Jα33 polyclonal antibody. Western blot and RT-PCR were used to detect Jα33 expression in MAIT cells of IBD mice. Successful molecular weight of about 38 kD fusion protein, obtained after immunization, a polyclonal rabbit antibody against the Jα33, Western blot results showed Jα33 polyclonal antibody can be detected in human peripheral blood mononuclear lymphocytes expressing Jα33, while in the HeLa No Jα33 expression; MAIT cells Jα33 expression in IBD mice intestinal tissue was significantly lower than the normal control. Polyclonal antibodies that recognize Jα33 in both human and mouse MAIT cells were successfully prepared and opened the door to studying the localization and function of MAIT cells.